Titin is the largest human protein, coded for by the largest human gene (TTN) and is highly expressed in the heart. A single titin polypeptide spans half the length of a sarcomere, connected to the Z disk and M lines at either end. In between, we find the I and A bands. The I band contains many folded repeat segments, acting as a molecular spring, permitting the sarcomere to expand and contract without deformation. The inextensible A band interacts with myosin thick filaments and plays a role in biomechanical signalling, though its role as a presumptive molecular ruler is under review (Granzier et al, 2014).

Although it was known for some time that TTN mutations were likely to play a role in cardiomyopathies (Siu et al, 1999), a comprehensive assessment of their significance was not possible until the advent of next generation sequencing technology (NGS), permitting the feasible and cost effective sequencing of this behemoth of a gene across a number of individuals. To put NGS in context, it took many laboratories around the world 10 years and over $1billion to first sequence the human genome using traditional Sanger sequencing; it can now be done in less than a week for $1000 in one place (Hayden 2014; International Human Genome Consortium 2001).

Dilated cardiomyopathy (DCM) is thought to have a familial or genetic basis in approximately 40% of cases. Whilst the two terms familial and genetic are not really synonymous, they had previously been used interchangeably in some quarters because it was felt that familial cases were likely to have a linked genetic cause, related to either a gene that had not yet been identified or a gene that could not be reliably interrogated. There were a large number of low frequency genes (found in approximately 1-2% of cases) linked to DCM (Hershberger et al, 2013). With the exception of LMNA (lamin) mutations (van Rijsingen et al, 2012), the identification of specific genetic mutations in DCM provided no added actionable clinical information for the patient in front of you in clinic (cascade screening is of course a different issue).

In 2012, Herman et al, published a landmark study which redefined our understanding of the genetic architecture of dilated cardiomyopathy (DCM). Using NGS, they identified the presence of truncating (radical, likely protein altering) mutations in TTN in up to a quarter of patients with idiopathic DCM.

Notable as this discovery was however, titin was not yet ready for primetime clinical cardiology. The paper left many questions unanswered, such as what the clinical significance of TTN mutations were, did the identification of a mutation change a patientís prognosis and how do you know which of the myriad of variants are significant? Furthermore, the paper found 3% of healthy normal individuals carried a truncating mutation in TTN, without any apparent consequences. This would imply that either TTN truncating mutations are not in fact that deleterious or that they have a variable and incompletely understood penetrance (penetrance refers to whether someone with the genetic mutation in question has any evidence of the disease phenotype; expressivity refers to how severe that phenotype is). In this context, 3% in the general population is a high number - for a genetic test to be of clinical use in a disease condition, there should be little chance that the result could be an incidental finding.

The publication of Roberts et al, in last monthís Science Translational Medicine, helps to address some of these issues.

They undertook a comprehensive analysis of the types of truncating mutations in patients with DCM. The original Herman paper demonstrated that many of the mutations in patients with DCM were clustered in the A band region, though it was unclear why this might be. Roberts et al found that A band exons are constitutively expressed, whereas I band exons are variably expressed. They show that the vast majority of pathogenic mutations (the mutations in DCM patients) were in highly expressed exons.

TTN is an enormous gene, capable of producing different protein isoforms, depending on the ways in which the different exons are kept together through differential mRNA processing, a process known as alternative splicing. The 2 major cardiac isoforms are a short stiff N2B isoform and a longer more complaint N2BA isoform. A different isoform is expressed in skeletal muscle. There are other shorter isoforms that are hardly expressed in cardiac muscle and arenít fully incorporated across the sarcomere. Roberts et al demonstrated that the TTN truncating mutations in normal individuals were enriched for I band mutations in exons with a low PSI (percentage spliced in), meaning exons that are not actually included in the final transcript. They also found that there was no difference in the total amount of titin protein in DCM patients with and without titin truncations, which suggests that the truncated protein is still incorporated into the sarcomere. We are yet to fully understand the mechanism of pathogenicity of the truncated protein.

Integrating all their studies across cohorts, they estimated that the discovery of a TTN truncating mutation that affects a highly expressed exon in a patient with DCM has a 93% probability of being pathogenic (likelihood ratio 14). Of course, this means that there remains a 7% chance that even a TTN truncating mutation that fits their revised criteria in a patient with DCM is not pathogenic.

Regarding clinical phenotype, of the A band mutations, the further along the protein towards the carboxyl terminus the truncation lies, the more severe the cardiac phenotype. Overall, DCM patients with TTN truncations had more severely impaired LV function, lower stroke volumes and thinner LV walls, as assessed by cardiac MRI. Notably, DCM patients with TTN truncations also had more non-sustained VT, even after correcting for LV ejection fraction. Their survival overall is reduced too, with a faster disease progression and earlier requirement for LVADs or transplants.

This detailed and rigorous study will significantly help researchers understand the significance of the genetic heterogeneity in TTN. There still remain approximately 1% of normal individuals with TTN truncating variants, and the factors that influence the variable penetrance of TTN truncating variants remain elusive. However, this work makes a strong case for the need for the stratified clinical management of DCM in the cardiology clinic.